The tick borne flaviviruses (TBFV) belong to the Family Flaviviridae, genus Flavivirus and comprise some of the most medically significant emerging and re-emerging viral pathogens. TBFV include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). TBFV are transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. Other flaviviruses include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the arthropod and the mammal) critical to virus replication and pathogenesis. We study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. Studies using LGTV will form the basis for work on the more virulent BSL-4 TBEV. 1. Analysis of virus interactions with the invertebrate host. Ixodid ticks represent the natural reservoir of TBFV, are critical for virus persistence in nature, and are the major vector for infection of humans. We have developed a custom Agilent microarray to investigate salivary gland transcriptional changes in Ixodes scapularis nymphs during feeding or after infection with LGTV. The first aim of this work is to identify tick proteins important during feeding. The second aim of this work is to identify tick proteins important for the replication or transmission of TBFV. Such genes may be targets for the development of novel vaccines or other technologies that have the potential to disrupt tick feeding and/or flavivirus transmission. Comparison of fed and unfed ticks revealed a dramatic metabolic change reflected by up-regulation of 578 transcripts during feeding. Of the 578 up-regulated genes, 128 are predicted to code for secreted proteins. These putative secreted proteins are interesting targets to pursue because they are likely present in the saliva and may facilitate feeding or evasion the host response. Interestingly, 135 of the 578 transcripts are classified as having no known function based on homology searches. These uncharacterized transcripts may have novel functions during feeding and are also interesting targets for further analysis. A clear temporal pattern of gene expression changes was observed showing clusters of genes were specifically up-regulated at 1, 2 or 3 days of feeding. Among the three days of feeding that were compared, the third day was the most unique. This may reflect the peak of metabolic activation in the tick when more salivary products are required to disarm the host response. The array data was validated using real-time PCR. Comparison of LGTV-infected and uninfected ticks has identified small, but significant gene expression changes associated with LGTV infection. We are currently validating the results of this microarray. One transcript that was significantly up-regulated in LGTV-infected ticks has homology to the lipocalin gene family. The putative histamine and serotonin-binding capability of lipocalins suggests they play a role in evasion of the host inflammatory and hemostatic responses, respectively. Thus, increased levels of lipocalin proteins may confer a survival advantage to the virus at the feeding site facilitating replication or transmission of LGTV. We are currently developing methods to introduce dsRNA into the tick to knock down this transcript and assess its role in LGTV replication and transmission. 2. Comparison of TBFV infection in vertebrate and invertebrate systems. TBFV are maintained in nature in an infectious cycle that involves both tick and vertebrate hosts. Thus, these viruses must successfully replicate in two very distinct systems. One basic difference between these two systems is that flavivirus infection of tick cells is persistent, whereas infection of mammalian cells tends to be acute and cytopathic. The genetic determinants underpinning these phenomena are not well understood. A. Viral determinants of pathogenesis in the arthropod vector and the mammalian host. In a previous study, we serially passaged LGTV in either tick or mouse cell lines and found a limited number of amino acid mutations in these passaged viruses compared to wild-type virus. Interestingly, the tick passaged virus exhibited reduced neuroinvasiveness when injected IP into mice compared to wild-type or the mouse passaged virus. Viral RNA from the brains of mice moribund after IP injection was characterized and five recurring amino acid changes were observed. Because of the location of these changes, we speculated that the amino acid mutations enabling neuroinvasiveness compensated for the mutations observed following passage in tick cells. We are utilizing a reverse genetics approach to introduce these amino acid changes individually or in combination into LGTV. These viruses will be rescued and tested in vivo to determine the role of specific amino acid changes in neuroinvasiveness. B. Comparison of TBFV infection in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. These rearranged membranes host virus replication and may protect replicative intermediates from intracellular antiviral systems. In the case of WNV and DEN, viral non-structural protein 4A (NS4A) has been implicated in the membrane rearrangements during infection. However, similar analysis has not been done with TBFV in tick cell lines. We are comparing virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal and electron microscopy. Electron microscopy has shown that TBFV-infected mammalian cells exhibit virus-induced vesicles, ER proliferation and accumulation of virions as early as 24 hrs post-infection. By 48 hrs post-infection, the virions assemble into paracrystalline arrays and large amounts of vesicles are observed. Significant cytopathic effect is observed at later timepoints and the cells begin to die. In the TBFV-infected tick cells, vesicle formation occurs later than in the mammalian cells. In addition, the vesicles have a different morphology appearing much more frequently as tubules in contrast to the round vesicles observed in the mammalian cells. No virions were detected in the tick cells at any time point post-infection. While this may simply be a consequence of the lower virus titer produced in the tick cells, these differences are currently being addressed using electron tomography to get a more detailed understanding of the virus-induced vesicle formation in the two different cell types. The results from the microscopy experiments will form the groundwork for biochemical and genetic investigations.